The aim of this study is to evaluate tropomyosin-4 (TM4) expression in infiltrating ductal breast carcinomas (IDCAs), as well as its prognostic significance. Using a 2-DE/MALDI-TOF mass spectrometry investigation coupled with an immunohistochemical approach, we have assessed the expression of TM4 in IDCAs, as well as in other types of breast tumors. Proteomic analyses revealed an increased expression of tropomyosin-4 in IDCA tumors. Using immunohistochemistry, overexpression of tropomyosin-4 was confirmed in 51 additional tumor specimens. Statistical analyses revealed, however, no significant correlations between tropomyosin-4 expression and clinicopathological parameters of the disease including tumor stage, patient age, estrogen and progesterone receptor status, and lymph node metastasis occurrence. A significant association was found, however, with a high Scarf-Bloom-Richardson (SBR) grade, a known marker of tumor severity. Additionally, the SBR component showing a correlation with TM4 expression was the tubular differentiation status. This study demonstrates the upregulation of tropomyosin-4 in IDCA tissues, which may highlight its involvement in breast cancer development. Our findings also support a link between tropomyosin-4 expression and aggressiveness of IDCA tumors.